1. Field of the Invention
This invention relates to a method of assaying urea and, more particularly, this invention relates to a colorimetric urea determination method and reagent especially suitable for assaying the urea concentration in body fluids.
2. Description of the Prior Art
Colorimetric methods of urea determination utilizing o-phthalaldehyde and a chromogenic compound are well known. Jung U.S. Pat. No. 3,890,099 (June 17, 1975) and Jung et al. "New Colorimetric Reaction for End Point, Continuous Flow, and Kinetic Measurement of Urea" (Clin. Chem., Vol. 21, No. 8 at 1136-40, 1975) describe a urea determination method and reagent wherein o-phthalaldehyde and a chromogenic compound are mixed with a urea-containing liquid sample.
The o-phthalaldehyde reacts with urea to produce a substantially colorless isoindoline derivative intermediate having one or both of two alternate structures.
The chromogenic compound, N-(1-naphthyl) ethylenediamine dihydrochloride, reacts with the intermediate to form a colored reaction product of unknown structure, the concentration of which is reportedly linearly related to the urea concentration of the sample and which follows Beer's law. The concentration of the colored substance is colorimetrically determinable at an absorbance maximum position of 505 nm. N-(1-naphthyl) ethylenediamine dihydrochloride has the following structure: ##STR1##
Denney U.S. Pat. No. 4,105,408 (Aug. 8, 1978), the details of which are hereby incorporated by reference, discloses five classes of chromogenic compounds which may be substituted for the chromogenic compound of the Jung disclosure. One of the classes of chromogenic compounds disclosed by Denney comprises 1, or 1,3 mono- or disubstituted hydroxy or methoxy naphthalene compounds having the following general structure: ##STR2## where R.sub.1 =--H or --CH.sub.3 and R.sub.2 =--H or --OCH.sub.3, or --OH. A preferred chromogenic compound of Denney is 1,3 dihydroxynaphthalene.
Each of the foregoing systems suffers from several disadvantages. According to Denney U.S. Pat. No. 4,105,408 (see col. 3, 1. 64-col. 4, 1. 21), the chromogenic compound of Jung is synthesized from .alpha.-naphthylamine, a known carcinogen, and therefore may contain at least trace amounts thereof. Furthermore, since the Jung reagents are conventionally stored in acidic solution, it is possible that the chromogen may decompose to form its carcinogenic precursor, .alpha.-naphthylamine. Also, the Jung reagents are reportedly interfered with by a variety of sulfa drugs, at least some of which are commonly present in body fluids subject to urea analysis.
Although the Denney reagents are not derived from a known carcinogen, several disadvantages are encountered with the Denney system. Firstly, it is believed that aqueous 1,3-naphthalene diols exhibit only limited stability in the presence of acid, rendering it impossible to store an acidic working reagent solution for an extended period of time. Unsubstituted 1,3-naphthalene diols are readily transported across all membranes, thereby increasing the risk of toxicity to laboratory personnel. Also, it is believed that 1,3-naphthalene diols may be interfered with to a significant extent by sulfa drugs and other drugs sometimes found in human body fluids.